Expression of hepatitis B S and preS2 proteins in methylotrophic

ABSTRACT

A process for the enhanced production of antigenic particles consisting essentially of hepatitis B S protein and preS 2  protein. Also disclosed are novel DNA molecules and hosts transformed with these molecules.

This is a divisional of application Ser. No. 07/193,174, filed on May13, 1988, now abandoned.

FIELD OF THE INVENTION

This invention relates to the field of recombinant DNA biotechnology. Inone aspect, this invention relates to a process for the enhancedexpression of antigenic particles consisting essentially of hepatitis BS protein and preS₂ protein in methylotrophic yeasts. In another aspectthe present invention relates to novel DNA molecules and novel yeaststrains transformed therewith.

BACKGROUND

Hepatitis B virus (HBV) causes both acute and chronic diseases and posesa worldwide public health problem. HBV manifests itself as a chronicallydebilitating infection which can result in progressively severe liverdamage, primary carcinoma and death. In the majority of cases, patientscompletely recover from HBV. However, a significant segment of thepopulation which is infected with HBV becomes chronic carriers of thedisease with the potential of transmitting the disease to others.

Recent advances in recombinant DNA techniques have provided many usefulmethods for elucidating the genetic structure of the HBV, as well asproviding the means for preparing vaccines against HBV. The HBV genomeis now known to consist of approximately 3.2 kilobase pairs of partiallydouble stranded DNA with a DNA polymerase covalently attached enclosedin a 27 nm nucleocapsid. The nucleocapsid is enveloped in a lipoproteincoat consisting of cellular lipids and hepatitis B surface antigens(HBsAg); this is called the virion and is 42 nm in diameter.

It has also been discovered that the vital coat consists of threedifferent but related surface proteins. These proteins are referred togenerally as S, PreS₂ and PreS₁ proteins. Each virion is comprised of300-400 S protein molecules and 40-80 preS₂ and pre-S₁ proteinmolecules.

The S protein consists of 226 amino acids and is the major component ofnormal vital lipoprotein coat. The S protein is approximately 24-25kilodalton (kDa), and may be referred to as P24 or P25. The S proteinmay also be glycosylated to a 27-28 kilodalton glyco-protein referred toas GP27 or GP28.

The second HBsAg protein is the PreS₂ surface antigen, also referred toas the middle HBsAg polypeptide. PreS₂ consists of 281 amino acidsformed by the addition of 55 amino acids to the N-terminus of the Sprotein. The PreS2 protein is approximately 31 kilodaltons and may bereferred to as the P31 protein. The PreS2 protein also has twoglycosylated forms, 33 kilodaltons and 36 kilodaltons, referred torespectively as GP33 and GP36. This antigen is thought to elicit anadditional antigenic response in persons who do not respond to S or whorespond weakly to S.

The third HBsAg protein is the PreS₁ surface antigen, also referred toas the late HBsAg polypeptide. PreS₁ consists of between 389-400 aminoacids (depending on the antigenic subtype of HBV). The sequence uniqueto PreS₁ consists of 108-119 amino acids which is added to theN-terminus of the complete PreS₂ protein. The PreS₁ protein isapproximately 43 kilodaltons and may also be referred to as the P43protein. PreS₁ also exists in a glycosylated form of 46 kilodaltonsdesignated as GP46 glycoprotein.

In the course of an HBV infection complete vital nucleocapsids areenveloped in a lipoprotein coat, forming 42 nm particles. Also formedduring the HBV infection are empty 22 nm particles which consist mostlyof the S and preS₂ proteins, and some preS₁ proteins. While the completevital nucleocapsid is infectious, the 22 nm empty particles are notinfections. The empty particles, however, will elicit an immune responsesufficient to confer immunity and may be used in the preparation ofvaccines to HBV.

Hepatitis B vaccines prepared with 22 nm particles historically wereprepared from the plasma of human carriers of HBV. Unfortunately 22 nmparticles derived from human plasma must be extensively purified toremove infectious HBV particles as well as any other plasma-bornepathogens. Additionally the preparation of hepatitis B vaccine bas beenseverely restricted because of the limited availability of human plasma.

Utilizing recombinant DNA biotechnology, it has been possible to expressthe hepatitis S protein in a 22 nm particle in transformed for examplemammalian cell lines, and Saccharomyces cerevisiae. The mammaliansystems currently utilized are expensive to use and the Saccharomycessystems produce relatively low yields of S protein.

Efforts to produce the antigenic and potentially more vaccine-effectivePreS₂ protein have proven unusually difficult. The PreS₂ protein hasbeen found to be very susceptible to proteolysis in recombinant systems.Proteolysis yields two smaller protein fragments which may not retainPreS₂ 's antigenicity. Additionally, the PreS₂ protein has been verydifficult to express in recombinant systems. The expression level ofPreS₂ is approximately 1/10th the level of the S protein produced in thesame recombinant systems.

It would be a significant contribution to the art to develop an enhancedprocess for the production of antigenic HBV particles containing the Sprotein and PreS₂ protein of HBV. These particles would combine themajor S protein with the more potentially antigenic PreS₂ protein in apotentially more vaccine-effective form.

Therefore, it is an object of this invention to provide a process forthe enhanced production of antigenic HBV particles consistingessentially of S protein and PreS₂ protein of HBV.

Yet another object of this invention is to provide novel vectorscontaining DNA sequences which code for S protein and PreS₂ protein.

A further object of this invention is to provide novel methylotrophicyeasts transformed with a vector or vectors capable of enhancedproduction of the HBV particle consisting of the S protein and anunglycosylated PreS₂ protein.

Still another object of this invention is the product produced by theprocess for the production of antigenic HBV particle consistingessentially of S protein and PreS₂ protein.

These and other objects of the invention will become apparent from thedisclosure and claims herein provided.

SUMMARY OF THE INVENTION

In accordance with the present invention, I have discovered a processfor the enhanced production of an antigenic HBV particle which comprisestransforming a methylotrophic yeast with at least one host-compatibleexpression cassette containing a structural gene for the S protein andat least one vector-compatible expression cassette containing astructural gene for the PreS₂ protein and culturing the resultanttransformants under conditions suitable to obtain the production ofparticles.

DETAILED DESCRIPTION OF THE FIGURES

FIG. 1 provides a representation of plasmid pA0801 which is apBR322-derived plasmid without an EcoRI site at position 1, and whichhas a BglII site in place of the pBR322 PvuII site, containing a 700 bpBglII/XhoI fragment of the 3' AOX1 termination sequence from pBSAGI5I(NRRL #18021).

FIG. 2 provides a representation of plasmid pA0802 which is a derivativeof plasmid pA0801 containing a promoter-gene-terminator expressioncassette from plasmid pBSAGI5I (NRRL #18021) inserted at the ClaI siteof plasmid pA0801.

FIG. 3 provides a representation of plasmid pA0803 which is a pA0802derived plasmid which has had the HBsAg coding sequence removed with aStuI-EcoRI digest and has an EcoRI site inserted at the same site.

FIG. 4 provides a representation of plasmid pA0811 which was derivedfrom plasmid pA0803, by digesting pA0803 with BamHI and inserting a 2.0kb fragment containing the Saccharomyces ARG4 gene.

FIG. 5A provides a schematic of the construction of plasmids pA0801 andpA0802.

FIG. 5B provides a schematic of the construction of plasmids pA0803 andpA0811.

FIG. 6 provides a representation of HBV which contains the PreS₂ gene ofHBV seratype adw. Plasmid AM6 is a derivative of the HBV genome shown inFIG. 6, wherein the BamHI-digested pBR322 plasmid is inserted at theBamHI site at position 26.

FIG. 7 provides a representation of plasmid pYM4, a pBR322-derivedplasmid containing the Pichia pastoris HIS4 gene inserted at the BamHIsite. Brackets indicate that site was destroyed. The HIS4 gene is ondeposit within pYJ30 (NNRL B-15890).

FIG. 8 provides a representation of plasmid pYM10. PYM10 is a derivativeof pYJ30 (NRRL B-15890) with the BamHI site at 2959 destroyed. Thebrackets in the Figure indicate a destroyed restriction site.

FIG. 9 provides a representation of plasmid pA0804 which contains alinear integrative site-specific vector in the fragment clockwise fromBglII to BglII. The structural gene may be inserted in the unique EcoRIsite of this plasmid.

DETAILED DESCRIPTION

The PreS₂ structural gene is well known in the art and has beensequenced by Lo, Characteristics of PreS₂ Region of Hepatitis B Virus,135 Biochemical and Biophysical Research Communications 382 (1986).Numerous workers in this field have cloned this structural gene andexpressed it such as Lo, and Valenzuela, Synthesis and Assembly in Yeastof Hepatitis B Surface Antigen Particles Containing the PolyalbuminReceptor, 3 Biotechnology 317 (1985) to name only two. The structuralgene may thus be obtained from workers in the field, synthesized orreisolated. It is also recognized that any PreS₂ seratype may be usedfor the practice of this invention.

The S structural gene is also well known in the art and has been alsosequenced by Lo, supra. The structural gene may be obtainedcommercially, synthesized or reisolated. It is also recognized that anyS seratype may be used for the practice of this invention.

The PreS₂ structural gene seratype adw used for one embodiment of thisinvention was obtained from plasmid AM6. Plasmid AM6 is a derivative ofthe HBV genome shown in FIG. 6, wherein the pBR322 plasmid is insertedat the BamHI site at position 26. The nucleotide sequence of this PreS₂structural gene is provided in Table 1. The plasmids used herein may becultured in any suitable E. coli host such as MC1061.

Two segments of PreS₂ structural genes were recovered from plasmid AM6and the nucleotide sequence for the first 13 amino acids of theN-terminus was synthesized in vitro. The synthesis of the nucleotidesequence used herein can be accomplished by either chemical or enzymaticmeans, such as chemical procedures based on phosphotriester, phosphiteor cyanoethylphosphoramidite chemistry.

The C-terminus coding region comprising 75% of the structural gene forPreS₂ was obtained from plasmid AM6 by a DraI digestion of the plasmid.The DraI digestion was performed using commercially available DraIendonuclease (all endonuclease were used following the manufacturer'srecommendation). The DraI fragments were then phenol extracted andethanol precipitated.

An octameric StuI linker (AAGGCCTT) was then prepared according tostandard DNA synthesis techniques and ligated to the DraI fragmentsusing T4 ligase in a blunt end ligation. The ligation was terminated byphenol extraction followed by ethanol precipitation. The resultingfragments were then digested with StuI endonuclease to remove multimersof StuI.

The StuI-linkered fragments were then digested with XbaI. The resultantStuI/XbaI fragment of approximately 600 bp containing the C-terminuscoding region of the PreS₂ structural gene was isolated by gelelectrophoresis.

This 600 bp fragment was then ligated with T4 ligase into a 5 kbXbaI/Stu digest of plasmid pYM4, FIG. 2, which had been isolated agarosegel electrophoresis.

The ligation mixture was then used directly to transform competent E.coli cells (MC1061), which were then grown in the presence ofampicillin.

                                      TABLE 1                                     __________________________________________________________________________     ##STR1##                                                                      ##STR2##                                                                      ##STR3##                                                                      ##STR4##                                                                      ##STR5##                                                                      ##STR6##                                                                      ##STR7##                                                                      ##STR8##                                                                      ##STR9##                                                                      ##STR10##                                                                     ##STR11##                                                                     ##STR12##                                                                     ##STR13##                                                                     ##STR14##                                                                     ##STR15##                                                                     ##STR16##                                                                     ##STR17##                                                                     ##STR18##                                                                     ##STR19##                                                                     ##STR20##                                                                     ##STR21##                                                                     ##STR22##                                                                    __________________________________________________________________________

Successfully transformed colonies were selected and the plasmid DNAextracted by the method of Birnboim and Doly Nucleic Acids Research7:1513 (1979)!

The extracted plasmid DNA was digested with StuI, phenol extracted andethanol precipitated. EcoRI linkers were prepared and ligated to theStuI fragments. Excess linkers were removed by EcoRI digestion. TheseEcoRI linkered fragments were further digested with XbaI andelectrophoresed to isolate the fragments containing the C-terminalportion of the preS₂ structural gene.

The XbaI-EcoRI digest was ligated into XbaI-EcoRI-cut pUC18. Theligation mixture was transformed into competent E. coli cells (MC1061),which was then grown in the presence of ampicillin. Transformed cellscontaining the C-terminal portion of the preS₂ structural gene wereselected by fragment digestion analysis employing ClaI and XbaI. Theplasmid selected by this process was designated pHS2-B.

The middle portion of the preS₂ gene was recovered by first digestingplasmid AM6 with XbaI and BamHI. The desired 250 bp fragment wasisolated by gel electrophoresis. The 250 bp XbaI/BamHI fragment was thenligated into pUC18 which had been digested with XbaI and BamHI and usedto transform E. coli MC1061. Cultures which grew in the presence ofampicillin were analyzed by recovering plasmid DNA and digesting it withBamHI. Those plasmids which contained a 2.7 Kb linear fragment uponelectrophoresis were deemed to have the desired 250 bp fragment. Onetransformed colony containing the 250 bp fragment was isolated and grownon a large scale. The plasmid DNA was isolated and purified from thecolony which was digested with EcoRI and BamHI. The vector band wasisolated by electrophoresis. The vector was then ligated with thefollowing kinased double stranded oligonucleotide. ##STR23##

The ligation reaction mixture was used to transform E. coli MC1061 andcolonies with the correct insert selected by ampicillin resistance. Thisplasmid was designated pPS2.

The N-terminus coding region of preS₂ was prepared as a syntheticoligonucleotide of the following sequence. ##STR24##

This sequence was cloned into a HindIII and PstI digest of pUC18. Thedesired transformants were characterized by the presence of anapproximately 75 bp fragment after EcoRI digestion and electrophoresis.The plasmids selected by the this method were designated pTBO-2A.

The middle portion of the gene was added by digesting the vector pTBO-2Awith PstI and XbaI; the insert was the 250 bp PstI-XbaI fragment frompPS2. Transformants were characterized by the presence of a >300 bpEcoRI fragment as well as 290 bp XbaI/HindIII fragment. The correctisolate was termed pTBO-3. The complete preS₂ gene was achieved byinserting the HindIII/XbaI fragment from pTBO-3 intoXbaI/HindIII-digested pHS2B. Ampicillin resistant transformants werecharacterized by the presence of a 825 bp EcoRI fragment. This constructwas termed pTBO4.

Culturing the E. coli strain listed above may be accomplished by anysuitable means. General techniques for culturing E. coli are alreadyknown in the art and any adaptation of these methods to the specificrequirements of the strains used herein is well within the abilities ofthose skilled in the art.

Recovery of plasmid DNA from E. coli can be accomplished by severaltechniques due to its compact size and closed spherical superhelicalform. For example following the harvest, host cells may be pelleted bycentrifugation and then resuspended and lysed. The lysate should becentrifuged to remove cell debris and the supernatant containing DNAretained. A phenol extraction can then be performed to remove most othercontaminants from the DNA. The phenol-extracted DNA may then be furthertreated using a density gradient centrifugation or gel filtrationtechnique to separate the plasmid DNA from the bacterial DNA. Thetechniques for achieving the separation alluded to above are well knownin the art and numerous methods of performing these techniques areknown.

Nuclease digestion of the plasmid may be accomplished by choosingappropriate endonucleases which will cut the selected plasmid in such away as to facilitate the recovery of the preS₂ structural gene. Theendonucleases used will depend on the plasmid from which the preS₂ geneis to be excised. For example, the preS₂ structural gene contained inplasmid AM6 could be recovered as described in Example I.

Gel electrophoresis of DNA may be accomplished using numerous techniquesknown in the art such as P. G. Sealy and E. M. Southern, GelElectrophoresis of Nucleic Acids--A Practical Approach (D. Rickwood andB. D. Hames, eds.) p. 39 (1982). Elution may also be accomplished usingnumerous techniques appropriate for the gel involved, such aselectroelution, diffusion, gel dissolution (agarose gels) or physicalextrusion (agarose gels). It is additionally recognized that elution maynot be necessary with some gels such as high-quality, low meltingtemperature agarose.

Once the fragment containing the preS₂ structural gene or fragmentsthereof is isolated, additional manipulations may be required before itis inserted in the vector. These manipulations may include, but are notlimited to the addition of linkers or blunt-ending the fragment.

For one embodiment of this invention the S gene was constructed from thepreS₂ gene by M13 mutagenesis. M13 mutagenesis was used to delete thefirst 165 base pairs (encoding the first 55 amino acids of the preS₂structural gene) resulting in the insertion of an EcoRI site immediately5' of the ATG start codon for the S structural gene. The techniques forM13 mutagenesis are well known to those skilled in the art, onerepresentative technique which could be used is the technique Zoller andSmith Methods in Enzymology 100:468(1983)!. M13 vectors a the reagentsneeded for mutagenesis are available from commercial sources such as NewEngland Biolabs.

Mutagenesis as described above was begun by first inserting the preS₂structural gene into the double stranded circular replicative form, orRF, of the M13 vector (for its ease of use, m13mp18 was selectedalthough other H13 vectors could have been used). The preS₂ structuralgene in plasmid pTB04 was propagated in E. colid MC1061, and the plasmidDNA was isolated utilizing the method of Birmboim and Doly, supra. ThepTB04 plasmid was then digested with EcoRI. The approximately 825 basepair EcoRI fragment containing the preS₂ structural gene was isolated bygel electrophoresis. This fragment was then inserted into the EcoRI siteof m13mp18. The ligation mixture was then used to transform competentbacterial cells such as JM101 or JM103. Transformants were selected onthe basis of a color reaction which indicated the desired fragment hadinterrupted the β-galactosidase gene and would form clear instead ofblue plaques on indicator plates.

The orientation of the insertion may be determined by sequencing,endonuclease digestion and gel electrophoresis, or any other suitabletechnique. One clone in which the initiator methionine had been insertedclose to the M13 universal primer was isolated and used for the firstmutagenesis.

Next, a short oligonucleotide was synthesized in vitro consisting of thefollowing nucleotide sequence:

    CGGGT-ACCGA-GCTCG-AATTC-ATGGA-GAACA-TCACA-TCAGG

The synthetic sequences used in the practice of this invention may beproduced by either enzymatic or chemical means. Suitable means includebut are not limited to chemical procedures based on phosphotriester,phosphite or cyanoethylphosphoramidite chemistry.

The single stranded form of N13 with the inserted structural gene wasprepared. The synthetic oligonucleotide which is partially complementaryto the 5' flanking region of the preS₂ gene and the 5' end of the Scoding region was then annealed to the single stranded M13 vectorcontaining the preS₂ structural gene. Using the partially complementarysynthetic oligonucleotide as a primer, DNA synthesis is carried out invitro with the Klenow fragment and deoxyoligonucleotide triphosphates at4° C. The partially complementary synthetic oligonucleotide was thenextended around the circular H13 template. The reaction six was used totransform competent JM101 or JM103 cells. The transformants werescreened by transferring the plaques to nitrocellulose and hybridizingwith a radioactively labeled oligonucleotide such that mutant strandshybridized, while the original template did not. These mutants were thenused to prepare a template, which was used to transform JM103. Thesetransformants were again screened as before. Positives from the secondscreening were then sequenced and plaques with the correct sequence wereidentified. The double stranded replicative form of these colonies wasthen digested with EcoRI and a 678 base pair fragment was isolatedcontaining the S structural gene. This sequence is provided in Table 2.

                                      TABLE 2                                     __________________________________________________________________________     ##STR25##                                                                     ##STR26##                                                                     ##STR27##                                                                     ##STR28##                                                                     ##STR29##                                                                     ##STR30##                                                                     ##STR31##                                                                     ##STR32##                                                                     ##STR33##                                                                     ##STR34##                                                                     ##STR35##                                                                     ##STR36##                                                                     ##STR37##                                                                     ##STR38##                                                                     ##STR39##                                                                     ##STR40##                                                                     ##STR41##                                                                     ##STR42##                                                                    __________________________________________________________________________

Following the isolation of the S and preS₂ structural genes, the genesare inserted into a suitable methylotrophic yeast vector such as aplasmid or linear site-specific integrative vector. Preferable vectorsfor the practice of this invention are those compatible with the Pichiagenus and most preferably Pichia pastoris.

Plasmids have long been one of the basic elements employed inrecombinant DNA technology. Plasmids are circular extrachromosomaldouble-stranded DNA found in microorganisms. Plasmids have been found tooccur in single or multiple copies per cell. Included in plasmid DNA isthe information required for plasmid reproduction, i.e. an origin ofreplication is included for bacterial replication. One or more means ofphenotypically selecting the plasmid in transformed cells may also beincluded in the information encoded in the plasmid. Phenotypic orselection markers, such as antibiotic resistance genes or genes whichcomplement defects in the host biochemical pathways, permit clones ofthe host cells which have been transformed to be recognized, selected,and maintained.

To express the preS₂ and the S structural gene in methylotrophic yeast,each gene must be operably linked to a 5' regulatory region and 3'termination sequence, which forms the expression cassette which will beinserted into the host via a vector.

The following terms are defined herein for the purpose of clarification.

Operably linked--refers to a juxtaposition wherein the components aconfigured so as to perform their function.

Regulatory region--DNA sequences which respond to various stimuli aaffect the rate of mRNA transcription.

3' Termination sequence--sequences 3' to the stop codon which functionto stabilize the mRNA such as sequences which elicit polyadenylation.

"Host compatible" refers to DNA sequences which will perform theirnormal function in hosts such as regulatory regions and 3' terminationsequences derived from hosts.

Preferred for the practice of the present invention are integrativevectors, such as the linear site-specific integrative vector of Cregg,as described in European Application Serial Number 86114700.7. Suchvectors comprise a said arranged sequence of at least 1) a firstinsertable DNA fragment; 2) a selectable marker gene; and 3) a secondinsertable DNA fragment.

Insertable DNA fragments are at least about 200 nucleotides in lengthand have nucleotide sequences which are homologous to portions of thegenomic DNA of the host. The various components of the linearsite-specific integrative vector are serially arranged forming a linearfragment of DNA such that the expression cassette and the selectablemarker gene are positioned between the 3' end of the first insertableDNA fragment and the 5' end of the second insertable DNA fragment. Thefirst and second insertable DNA fragments are oriented with respect toone another in the serially arranged linear fragment as they are sooriented in the parent genome.

Nucleotide sequences useful as the first and second insertable DNAfragments are nucleotide sequences which are homologous with separateportions of the native genomic site at which genomic modification is tooccur. Thus, for example, if genomic modification is to occur at thelocus of the alcohol oxidase gene, the first and second insertable DNAfragments employed will be sequences homologous with separate portionsof the alcohol oxidase gene locus. For genomic modification inaccordance with the present invention to occur, the two insertable DNAfragments must be oriented with respect to one another in the linearfragment in the same relative orientation as they exist in the parentgenome. Examples of nucleotide sequences which could be used as firstand second insertable DNA fragments are nucleotide sequences selectedfrom the group consisting of the alcohol oxidase (AOX1) gene,dihydroxyacetone syntha (DHAS1) gene, p40 gene and HIS4 gene. The AOX1gene DHAS1 gene, p40 gene and HIS4 gene are disclosed in Europeanapplication 85113737.2 filed Oct. 29, 1985, incorporated herein byreference.

The first insertable DNA fragment may contain an operable regulatoryregion which my comprise the regulatory region utilized in theexpression cassette. The use of the first insertable DNA fragment as theregulatory region for an expression cassette is a preferred embodimentof this invention. FIG. 4 provides a diagram of a vector utilizing thefirst insertable DNA fragment as a regulatory region for a cassette.

Optionally as shown in FIG. 9 an insertion site or sites and atermination sequence may be placed immediately 3' to the firstinsertable fragment. This conformation of the linear site-specificintegrative vector has the additional advantage of providing a readysite for insertion of a structural gene without necessitating theaddition of a compatible 3' termination sequence.

It is also necessary to include at least one selectable marker gene inthe DNA used to transform the host strain. This facilitates selectionand isolation of those organisms which have incorporated thetransforming DNA. The marker gene confers a phenotypic trait to thetransformed organism which the host did not have, e.g., restoration ofthe ability to produce a specific amino acid where the untransformedhost strain has a defect in the specific amino acid biosynthetic pathwayor resistance to antibiotics and the like.

Exemplary selectable marker genes may be selected from the groupconsisting of the HIS4 gene and the ARG4 gene from Pichia pastoris andSaccharomyces cerevisiae, the invertase gene (SUC2) from Saccharomycescerevisiae, or the neomycin phosphotransferase gene from the E. colitransposable elements Tn601 or Tn903.

Those of skill in the art recognize that additional DNA sequences canalso be incorporated into the vectors employed in the practice of thepresent invention, such as for example, bacterial plasmid DNA,bacteriophage DNA, and the like. Such sequences enable the amplificationand maintenance of these vectors in bacterial hosts.

If the first insertable DNA fragment does not contain a regulatoryregion, a suitable regulatory region will need to be inserted operablylinked to the structural gene, in order to provide an operableexpression cassette. Similarly if no 3' termination sequence is providedat the insertion site to complete the expression cassette, a 3'termination sequence will have to be operably linked to the structuralgene to be inserted.

Those skilled in the art are aware of numerous regulatory regions whichhave been characterized and could be employed in conjunction withmethylotrophic yeasts. Exemplary regulatory regions include but are notlimited to yeast regulatory regions selected from the group consistingof acid phosphatase, galactokinase, alcohol dehydrogenase, cytochrome c,alpha-mating factor and glyceraldehyde 3-phosphate dehydrogenaseregulatory regions isolated from Saccharomyces cerevisiae; the primaryalcohol oxidase (AOX1), dihydroxyacetone synthase (DHAS1), the p40regulatory regions, and the HIS4 regulatory region derived from Pichiapastoris and the like. Presently preferred regulatory regions employedin the practice of the present invention are those characterized bytheir ability to respond to methanol-containing media, such regulatoryregions selected from the group consisting of AOX1, DHAS1, p40 anddisclosed in European Application 85113737.2 filed Oct. 29, 1985.

The most preferred regulatory region for the practice of this inventionis the AOX1 regulatory region.

3' termination sequences may be utilized in the expression cassette orbe part of the vector as discussed above. 3' termination sequences mayfunction to terminate, polyadenylate and/or stabilize the messenger RNAcoded for by the structural gene when operably linked to a gene. A fewexamples of illustrative sources for 3' termination sequences for thepractice of this invention include but are not limited to theSaccharomyces cerevisiae, Hansenula polymorpha, and Pichia 3'termination sequences. Preferred are those derived from Pichia pastorissuch as those selected from the group consisting of the 3' terminationsequences of AOX1 gene, DHAS1 gene, p40 gene and HIS4 gene. Particularlypreferred is the 3' termination sequence of the AOX1 gene.

Those of skill in the art recognize that additional DNA sequences canalso be incorporated into the vectors employed in the practice of thepresent invention, such as for example, bacterial plasmid DNA,bacteriophage DNA, and the like. Such sequences enable the amplificationand maintenance of these vectors in bacterial hosts.

Another suitable vector would be an integrative vector which wouldcomprise an arranged sequence of at least 1) an insertable DNA fragment,2) a selectable marker gene, 3) an expression cassette and optionally 4)another insertable DNA fragment. The components of the integrativevector are equivalent to those used in the linear integrativesite-specific vector except there needs to be only one insertable DNAfragment, however integrative vectors are believed to integrate theentire vector by homologous recombination. Preferred are circularintegrative vectors such as is shown in FIG. 4. For the practice of thecurrent invention it is currently preferred to use linear transformationvectors such as the BglII fragments of the constructs shown in FIG. 9and the circular form of the integrative vector in FIG. 4.

The insertion of a S or preS₂ structural gene into suitable vectors maybe accomplished by any suitable technique which cleaves the vectorchosen at an appropriate site or sites and results in at least oneoperable expression cassette containing a S or preS₂ structural genebeing present in the vector.

Ligation of a S or preS₂ structural gene may be accomplished by anyappropriate ligation technique such as utilizing T4 DNA ligase.

The initial selection, propagation, and optional amplification of theligation mixture of a S or preS₂ structural gene and a vector ispreferably performed by transforming the mixture into a bacterial hostsuch as E. coli. Suitable transformation techniques for E. coli are wellknown in the art. Additionally, selection markers and bacterial originsof replication necessary for the maintenance of a vector in a bacterialhost are also well known in the art.

The isolation and/or purification of the desired plasmid containing a Sor preS₂ structural gene in an expression system may be accomplished byany suitable means for the separation of plasmid DNA from the host DNA.

Similarly the vectors formed by ligation may be tested preferably afterpropagation to verify the presence of a S or preS₂ gene and its operablelinkage to a regulatory region and a 3' termination sequence. This maybe accomplished by a variety of techniques including but not limited toendonuclease digestion, gel electrophoresis, or endonucleasedigestion-Southern hybridization.

For the practice of this invention at least two different compatibleexpression cassettes must be transformed into the host cell. There areseveral methods by which these two different expression cassettes may beinserted into the host such as placing two functional expressioncassettes in a vector (vital, plasmid or linear site-specificintegrative) and transforming a host with these vectors. Another methodof transforming a host with at least two different expression cassettes(S and preS₂), would be to transform the host with two differentvectors, one containing the S expression cassette and the othercontaining the preS₂ expression cassette. The transformation with twodifferent vectors (dual transformation) could be accomplished bysimultaneous transformation (both vectors present in a singletransformation event) or sequentially (one vector transformed into thehost followed by a second transformation with the other vector in to thepreviously transformed host cells). Dual sequential transformation iscurrently the preferred method of transformation.

Transformation of plasmids or linear vectors into yeast hosts may beaccomplished by suitable transformation techniques including but notlimited to those taught by Hinnen et al, Proc. Natl. Acad. Sci. 75,(1978) 1929; Ito et al, J. Bacteriol 153, (1983) 163; Cregg et al Mol.Cell Biol. 5 (1985) pg. 3376; or Sreekrishna et al, Gene, 59 (1987) pg.115. Preferable for the practice of this invention is the transformationtechnique of Cregg. It is desirable in one embodiment of this inventionto utilize an excess of linear vectors and select for multipleinsertions by Southern hybridization.

The yeast host for transformation may be any suitable methylotrophicyeast. Methylotrophic yeast include but are not limited to yeast capableof growth on methanol selected from the genera consisting of Hansenula,Candida, Kloeckera, Pichia, Saccharomyces, Torulopsis and Rhodotorula. Alist of specific species which are exemplary of this class of yeasts maybe found in C. Anthony, The Biochemistry of Methylotrophs, 269 (1982).Presently preferred are methylotrophic yeasts of the genus Pichia suchas the auxotrophic Pichia pastoris GS115 (NRRL Y-15851) or PPF1(NRRL-Y-18017). Auxotrophic methylotrophic yeasts are also advantageousto the practice of this invention for their ease of selection. It isrecognized that wild type methylotrophic yeast strains may be employedwith equal success if a suitable transforming marker gene is selected,such as the use of SUC2 to transform Pichia pastoris to a strain capableof growth on sucrose or an antibiotic resistance marker is employed,such as G418^(R) gene.

For the practice of the current invention it is currently preferred touse the two selection markers in combination, to select for the stabletransformation of S and preS₂ into the host. Thus it is advantageous forthe practice of the invention to use host and vector combinations whichassure that when two different selection markers are used in twodifferent vectors each marker may be independently selected for. Onesuch host and vector combination would be the use of PPF1 (HIS4, ARG4)with vectors pA0804 (HIS4 marker) and pA0811 (ARG4 marker). Anotherpossible combination would be the use of an auxotroph defective in onlyone pathway in combination with a gene which is complementary to thedefect and an antibotic resistance gene or a gene which generates a newphenotype such as SUC2.

Transformed methylotrophic yeast cells can be selected for usingappropriate techniques including but not limited to culturing previouslyauxotrophic cells after transformation in the absence of a biochemicalproduct required (due to the cell's auxotrophy), selection by thedetection of a new phenotype ("methanol slow"), or culturing in thepresence of an antibiotic which is toxic to the yeast in the absence ofa resistance gene contained in the transformant.

Isolated transformed methylotrophic yeast cells are cultured byappropriate fermentation techniques such as shake flask fermentation,high density fermentation or the techniques disclosed by Cregg et al,High-Level Expression and Efficient Assembly of Hepatitis B SurfaceAntigen in the Methylotrophic Yeast, Pichia Pastoris, 5 Bio/Technology479 (1987).

Expression may be accomplished by methods appropriate to the regulatoryregion employed. Preferably if methanol responsive regulatory regionsare utilized, the induction of expression may be accomplished byexposing the transformed cells in a nutrient media to an appropriatealcohol for the regulatory region employed.

The antigenic particles may be recovered in a crude form by lysingtransformed cells which have been induced for a sufficient period, usingstandard techniques such as bead milling, followed by centrifugationsufficient to remove cellular debris. Those of skill in the art areaware of numerous methods available for the extraction of a heterologousprotein from unicellular host organisms which could be substituted forthe general extraction technique above or for further purification. Thepreferred method of purification for the practice of this invention isdisclosed in an application filed April 13, 1988 by Cregg et al.,attorney docket number 32342US incorporated herein by reference.

The following non-limiting examples are provided to further illustratethe practice of this invention.

EXAMPLES

General information pertinent to the Examples:

Strains

Pichia pastoris GS115 (his4) NRRL Y-15851 was the host yeast strain usedin these examples.

Pichia pastoris PPF1 (arg4 his4) NRRL Y-18017.

E. coli MC1061 NRRL 18016.

E. coli JM103 delta (lac pro) thi rpsL (strA) supE endA sbcB hsdR.

Media

    ______________________________________                                        YPD, 1 liter    20 g yeast extract                                                            20 g peptone                                                                  20 g dextrose                                                 LB broth, 1 liter                                                                             5.0 g yeast extract (Difco)                                                   10.0 g tryptone (Difco)                                                       5.0 g NaCl                                                    TE buffer       1.0 m M EDTA                                                                  in 0.01 M (pH 7.4) Tris buffer                                PEG Solution    20% polyethylene glycol-3350                                                  10 m M CaCl.sub.2                                                             10 m M Tris-HCl (pH 7.4)                                                      filter sterilize                                              Solution A      0.2M Tris-HCl (pH 7.5)                                                        0.1M MgCl.sub.2                                                               0.5M NaCl                                                                     0.01M dithiothreitol (DTT)                                    Solution B      0.2M Tris-HCl (pH 7.5)                                                        0.1M MgCl.sub.2                                                               0.1M DTT                                                      20X SSPE        4.4 g NaOH                                                                    7.4 g Na.sub.2 EDTA                                                           27.6 g NaH.sub.2 PO.sub.4.H.sub.2 O                                           210 g NaCl                                                                    pH adjusted to 7.5-8.0 with NaOH                                              H.sub.2 O to 1 liter                                          10X Transfer Buffer                                                                           5.0 g NaCl                                                                    96.8 g Trizma Base                                                            9.74 g glycine                                                                water to 1 liter                                              ______________________________________                                    

Example I Construction of Vector pTB04

Plasmid AM6 is a derivative of the HBV genome shown in FIG. 6, whereinthe pBR322 plasmid is inserted at the BamHI site at position 26.

The vector pTB04 contains the gene encoding the 281 amino acid preS₂form of the hepatitis B surface antigen. The gene was constructed inthree segments: the C-terminal 75% of the structural gene, an N-terminallinker encoding 13 amino acids, and the remaining central portion. Theplasmid AM6 containing the preS₂ gene, adw seratype, was the source ofthe C-terminal portion and the middle portion of the preS₂ gene usedhere. The sequence is disclosed in Valenzuela et al., ICN-UCLA Symposiaon Animal Virus Genetics, p. 57-70 (1980), with the followingmodification: ##STR43## A. C-terminal portion of preS₂

(All restriction enzymes were obtained from Boehringer Mannheim, andused according to manufacturer's instructions).

The C-terminal portion was isolated from the plasmid AM6 (FIG. 1) bydigestion with DraI, which cuts the HBV genome at two places, one ofwhich is at the codon for the last amino acid of the surface antigen.The ends were dephosphorylated by treatment with calf intestinalalkaline phosphatase in a 30 μl reaction volume (1U enzyme at 37° C. for1 hour in 50 mM Tris.Cl, pH 9.0, 1 mM MgCl₂, 100 μM ZnCl₂, 1 mMspermidine). The entire digest was phenol extracted and ethanolprecipitated (Maniatis et al.). An octameric StuI linker (AAGGCCTT) wassynthesized by a DNA synthesizer from Applied Biosystem Model 380A usingcyanoethylphosphoramidite chemistry. 1 μg of StuI linkers was dissolvedin distilled water. A 10 ng aliquot was removed and labeled withphosphate in a 50 μl total volume containing 70 nM Tris.Cl, pH 7.6, 10nM MgCl₂, 5 μM dithiothreitol, 1 nM ATP and 10 units of polynucleotidekinase for 30 minutes at 37° C. The linkers were heated to 90° C. toterminate the enzymatic reaction, and slow-cooled to room temperature tofacilitate double stranded DNA formation. The StuI linkers were added tothe DraI digest above and ligated with T4 ligase as follows. Thereaction occurred in a 10 μl volume containing 6.6M Tris.Cl, pH 7.6, 5mM MgCl₂, dithiothrietol, 1 mM ATP and 1 Weiss unit of T4 ligase for 1hour at 23° C. The ligation reaction was terminated by phenol extractionfollowed by ethanol precipitation. A StuI restriction digest was thenperformed with >50 U of enzyme overnight to remove multimers of the StuIlinker. The combination of DraI digestion and StuI linkers restored thetranslation stop codon removed by DraI digestion.

The StuI-linkered DraI fragments were digested with XbaI, which yieldedthe desired StuIbaI fragment of approximately 600 bp; it was isolatedfrom a 0.8% preparative agarose gel. This fragment contained theC-terminal 75% of the gene and was cloned into the vector pYM4 (FIG. 2)which had been digested with XbaI and StuI, and dephosphorylated asabove. (pYM4 can be obtained from plasmid pYM30 by digesting pYM30 withClaI and re-ligating the ends. pYM30 is available in an E. coli hostdeposited at the Northern Regional Research Center, United StatesDepartment of Agriculture, Peoria, Ill., accession number NRRL B-15890).The 5.7 Kb restriction fragment of pYM4 was isolated from a 0.8%preparative agarose gel. Vector pYM4 was employed solely for itsconvenient restriction sites. 50 ng of the vector and 500 ng of theinsert were ligated at 23° C. for 1 hour in 50 mM Tris HCl pH 7.4, 10 mMHgCl₂, 10 mM dithiothreitol, 1 nM spermidine, 1 mM ATP with 1 Weiss Unitof T4 ligase in a 10 μl volume. The ligation reaction was used directlyto transform competent MC1061 cells (E. coli) to ampicillin resistance.E. coli strain MC1061 is available at the Northern Regional ResearchCenter, United States Department of Agriculture, Peoria, Ill., accessionnumber NRRL-18016. MC1061 has the following genotype: F(-), ara D139delta (lacIPOZY) X74 galk galU hsr hsm(+) rpsL delta (araABOIC leu)7697. MC1061 was rendered for transformation in the following manner. Amid-log culture (50 ml) of E. coli MC1061 was harvested bycentrifugation in a Damon IEC DPR600 at 3,000 rpm for 5 min. at 4° C.and washed in 10 nM NaCl. The culture was resuspended in 25 ml of 50 mMCaCl₂ for 30 minutes at 0° C. The cells were centrifuged as above andresuspended in 2 ml of 50 mM CaCl₂. For transformation, the ligationreaction was added to 100 μl of the competent cell suspension andincubated at 0° C. on ice for 15 minutes, heat shocked at 37° C. for 5minutes and incubated at 23° C. for 5 minutes. The cells were plateddirectly onto LB agar plates containing 50 μg/ml ampicillin. The plateswere incubated at 37° C. for 10-16 hours. The resulting colonies wereharvested and characterized by restriction digestion. Cells were grownin 5 ml of L-broth containing 50 μg/ml ampicillin for 5 hr. at 37° C.and DNA was prepared by the method of Birnboim and Doly Nucleic AcidsResearch 7:1513 (1979)!. The minipreps were digested with BamHI andXbaI. Cultures yielding a 1.5 Kb Xba/Bam fragmented were deemed to havethe insert and a large scale DNA prep of one culture was purified asabove, followed by banding on a cesium chloride-ethidium bromidegradient. This clone was called pHS1.

The plasmid pHS1 was digested with Stu1, dephosphorylated as above,phenol extracted and ethanol precipitated. EcoRI linkers (GGAATTCC)synthesized as above were phosphorylated, self annealed, and ligated tothis blunt ended DNA. Excess linkers were removed by overnight EcoRIdigestion and the DNA was subsequently digested with XbaI followingphenol extraction and ethanol precipitation. A doublet containing the600 bp XbaI-EcoRI fragment of interest and a vector fragment of 582 bp(XbaI-EcoRI) was isolated by 1.0% preparative gel electrophoresis. Thesefragments (500 ng) were ligated into XbaI-EcoRI-digested anddephosphorylated pUC18 (50 ng) as described above and used to transformMC1061 to ampicillin resistance as described above. Restriction digestsof miniprep DNA were used to determine which of the two fragments hadbeen cloned. The undesirable fragment had a ClaI site such that aClaI/XbaI double digest would yield fragments of approximately 560bp+2400 bp, whereas the correct fragment yielded a linear 3 Kb fragmentupon ClaI/Xb digestion. Candidates were digested with EcoRI and XbaI toyield fragments 600 bp+2400 bp. One such clone was purified on largescale and termed pHS2-B. This contains an EcoRI site after the lastcodon at the C-terminal region of the complete preS₂ gene.

B. Middle Portion of preS₂ Gene

The middle portion of the preS₂ gene was cloned as follows. The plasmidAM6 was digested with XbaI and BamHI and a fragment of 250 bp wasisolated from a 0.8% preparative agarose gel. This fragment (50 ng) wasligated as described above to 50 ng of pUC18 digested with XbaI andBamHI and dephosphorylated. The ligation reaction was used to transformE. coli strain MC1061 to ampicillin resistance as above. Minipreps weredigested with BamHI and those containing a 2.7 Kb linear fragment werechosen. One isolate was grown on large scale and DNA was isolated andpurified as described. This clone is called pPS1. The clone was cut withEcoRI and BamHI and the vector band isolated and purified by 0.8%preparative agarose gel electrophoresis. To this vector was ligated thefollowing kinased double stranded oligonucleotide synthesized as above:##STR44## The ligation reaction was used to transform E. coli MC1061 toampicillin resistance. Minipreps were characterized by PstI digestion.One clone containing a 250 bp PstI fragment was chosen, a large scaleDNA prep was performed, and the plasmid pPS2 was isolated.

C. N-terminal Portion of preS₂ Gene

The N-terminal region encompassing the EcoRI linker and the codingsequences for the first thirteen amino acids was generated from asynthetic oligonucleotide containing the following sequence synthesizedas above: ##STR45## This fragment contained HindIII and PstI ends aswell as an EcoRI sequence preceding the ATG. This sequence was clonedinto HindIII- and PstI-digested and dephosphorylated pUC18 by ligating aten-fold excess of the oligo into the vector and characterizing thetransformants by the presence of a small EcoRI site (˜75 bp). Such aclone was designated pTBO-2A.

The middle portion of the gene was added by digesting the vector pTBO-2Awith PstI and XbaI; the insert was the 250 bp Pst-Xba fragment frompPS2. Transformants were characterized by the presence of a >300 bpEcoRI fragment as well as 290 bp XbaI/HindIII fragment. The correctisolate was termed pTB0-3. The complete preS₂ gene was achieved byinserting the HindIII/XbaI fragment from pTB0-3 intoXbaI/HindIII-digested pHS2B. Ampicillin resistant transformants werecharacterized as above by the presence of a 825 bp EcoRI fragment. Thisconstruct was termed pTB04.

Example II Construction of Vector pTB05A

A vector containing the gene coding for preS₂ was constructed fromvectors pA0804 and pTB04 (Examples V and I, respectively). 2 μg ofpA0804 was digested with EcoRI as before and treated with alkalinephosphatase in a 30 μl reaction volume (1 U enzyme at 37° C. for 1 hourin 50 mM Tris.Cl pH 9.0, 1 mM MgCl ₂, ZnCl₂, 1 mM spermidine). pTB04 wassubjected to EcoRI digestion and an 825 bp fragment encoding the preS₂gene was released. This fragment was purified using preparative agarosegel electrophoresis using 0.8% agarose. 500 ng of the fragment and 50 ngof pA0804 were ligated using methods described in Example I. Theresulting vector was used to transform MC1061 to ampicillin resistanceusing the method described in Example I. The DNA was isolated using themethod of Birnboim and Doly Nucleic Acids Research 7:1513 (1979)! andcharacterized by digestion with PstI. A clone containing a 2.1 Kb PstIfragment was determined to have the insert in the correct orientationand was designated pTB05A.

Example III Construction of pTB047 Template

1 μg of double stranded m13mp18 DNA (from Example I) was digested withEcoRI and dephosphorylated by treatment with calf intestinal alkalinephosphatase in a 30 μl reaction volume (1 U enzyme at 37° C. for 1 hourin 50 mM Tris.Cl, pH 9.0, 1 mM MgCl₂, 100 mM ZnCl₂, 1 mM spermidine).The 825 bp EcoRI fragment containing the preS₂ gene was isolated frompTB04 (see Example I) by digestion with EcoR1, and was then isolatedfrom a 0.8% preparative agarose gel. 50 ng of m13mp18 vector and 500 ngof the EcoRI insert were ligated with T4 DNA ligase as follows. Thereaction occurred in a 10 μl volume containing 6.6M Tris.Cl, pH 7.6, 5mM MgCl₂, 5 mM dithiothrietol, 1 mM ATP and 1 Weiss unit of T4 ligasefor 1 hour at 23° C.

The ligation mixture was then used to transform E. coli JM103 cellswhich bad been made competent in the following manner. A mid-log culture(50 ml) of E. coli JM103 cells was harvested by centrifugation in aDamon IEC DPR600 clinical centrifuge at 3,000 rpm for 5 min. at 4° C.and washed in 10 mM NaCl. The culture was resuspended in 25 ml of 50 mMCaCl₂ for 30 min. at 0° C. The cells were centrifuged as above andresuspended in 2 ml of 50 mM CaCl₂.

For transformation, the ligation reaction was added to 100 μl of thecompetent cell suspension and incubated at 0° C. on ice for 15 minutes,beat shocked at 37° C. for 5 minutes, and incubated at 23° C. for 5minutes. The cells were then plated in soft agar containing IPTG andX-gal and spread on to LB media, and incubated at 37° C. overnight, andthe plates were screened for clear plaques.

In order to determine which plaques had the insert in the correctorientation, double-stranded DNA was prepared and separate digests withEcoRI and XbaI were performed. One was found to have the initiatormethionine of the insert close to the M13 universal primer, and wouldthen generate a template containing the anti-sense strand of the insert.This was designated pTB047.

Example IV Construction of pTBO-6 and pHB6

A DNA sequence encoding a 226 amino acid form of HBsAg (the S form) wascreated by deleting the 165 bp encoding the first 55 amino acids ofpreS₂. This was accomplished by subjecting the template pTB047 (fromExample III) to M13 primer-directed deletion mutagenesis using thefollowing oligonucleotide primer: ##STR46## This was synthesized usingan Applied Biosystems DNA Synthesizer Model 380A usingcyanoethylphosphoramidite chemistry. Mutagenesis was performed accordingto the following.

A large scale miniprep was performed on positive plaques which had beenincubated for approximately 7 hours in 2 mls of LB media. 25 mls of LBmedia was inoculated with 250 μl of freshly grown JM103 cells. Theculture was grown for 1 hour and inoculated with 100 μl of the 7 hourold plaque culture. The culture was then grown overnight. The culturewas centrifuged twice at 10,000 rpms for 10 minutes on a Sorvall RC-5Brotor SS34 to clear the supernatant. 3.5 ml of 20% PEG/2.5M NaCl wasadded to the culture and it was incubated for 5 hours at 4° C. Theculture was then centrifuged as above for 10 minutes. The supernatantwas discarded and the pellet was resuspended in 2 mls of TE buffer. Thepellet was then extracted with phenol (equilibrated with TE), extractedonce with phenol/chloroform, extracted twice with CHCl₃ and once withether. 8M LiCl was added to attain a final concentration of 0.8M. 3volumes of ethanol were added and the solution left overnight at 20° C.to precipitate the DNA present. The solution was next centrifuged for10,000 rpms for 10 minutes as previously described and rinsed with 70%ethanol. The precipitate was resuspended in 150 μl of 10 mM Tris.Cl, pH7.4.

One pmole of M13 recombinant template was mixed with 20 pmole of theoligonucleotide primer, and 1 μl of solution A. dH₂ O was added to givea final volume of 10 μl. The sample was incubated at 65° C. for 5minutes, and the temperature was then reduced to 37° C. for 30 minutes.

The following was then added to the sample:

    ______________________________________                                               Solution B     1     μl                                                    10 mM dATP     1     μl                                                    10 mM dCTP     1     μl                                                    10 mM dGTP     1     μl                                                    10 mM dTTP     1     μl                                                    5 u/μl Klenow                                                                             2     μl                                                    dH.sub.2 O     3     μl                                                                   20    μl                                             ______________________________________                                    

and allowed to incubate at 15° C. for at least 4-6 hours.

The sample was then diluted 1:40 with dH₂ O. 5 μl was used to transform6 tubes of competent JM103 cells (200 μl each). The transformed JM103cells were plated on LB media in a soft agar overly. The positiveplaques were then screened by filter hybridization. A hybridizationprobe complementary to the oligonucleotide primer was synthesized asdescribed above. 15 pmole of this probe was incubated at 65° C. for 10minutes in a total volume of 25 μl. 3 μl of 10× kinase buffer (Maniatiset al.), 1 μl 10 mM ATP, and 1 μl polynucleotide kinase (100 U/μl) wereadded. The sample was incubated for 1 hour at 37° C. and run over a G50Sephadex column. The first peak off the column was collected.

Nitrocellulose filters were prepared for hybridization with the aboveprobe by placing and orienting the filters on the transformation platesfor 5-10 minutes. The filters were then removed from the plates andfloated on a denaturing solution (1.5M NaCl, 0.5N NaOH) for 3 minuteswith the backside on top of the solution. The filters were submerged inthe denaturing solution for 5 minutes, and then transferred to aneutralizing solution (1M Tris.Cl, pH 8, 1.5M NaCl) for 5 minutes. Theneutralized filter was then transferred to 2×SSC (1×SSC is 150 mM NaCl,15mM NaCitrate) for 5 minutes. The filter was air dried and baked for 1hour at 80° C. under a vacuum. The filters were prehybridized for 1 hourat 65° C. in a sealed plastic bag containing 5 ml of hybridizationbuffer, 10× Denhardts (1× Denhardts is 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin) 0.5% SDS, and 5×SSPE. Thebuffer was replaced with 5 ml/filter of fresh hybridization buffer. Theradioactive complementary oligonucleotide previously prepared was firstincubated at 65° C. for 5 minutes, and then enough probe was added tothe fresh hybridization buffer containing the filter to give 1×10⁶cpm/ml. Hybridization was performed at 5° C. below the calculatedmelting temperature of the probe for 4 hours.

The filters were then washed three times for 10 minutes each with 6×SSCat room temperature. The filters were finally washed one time with 6×SSCat the hybridization temperature. The filters were placed on a 3 MMWhatman paper to dry, and then exposed to film (marked for orientation)overnight. Three positive plaques were each picked and grown separatelyin 2 mls of LB broth at 37° C. for 5 hours.

Mini template preps were performed on each of these positive plaques.One ml of the plaque culture was transferred into an Eppendorf tube andcentrifuged for 5 minutes in an Eppendorf Model 5414 Centrifuge. 800 μlof the supernatant was recovered and 200 μl of 200% PEG 2.5M NaCl addedthereto. The supernatant was then incubated at room temperature for 10minutes, and centrifuged for 10 minutes in the Eppendorf centrifuge. Thesupernatant was removed by aspiration and the pellet was redissolved in200 μl TE (10 ml Tris, pH 7.4; 1 mM ETDA). The redissolved pellet wasthen phenol/chloroform extracted and the template DNA in the upperaqueous phase was precipitated by the addition of a LiCl solution unitla 0.8M concentration of LiCl was reached. 21/2-3 volumes of ethanol wasadded and the sample was precipitated on dry ice for 5 minutes. Theprecipitate was centrifuged for 10 minutes as described above. The finalvolume was brought up to 150 μl TE.

200 μl of competent JM103 cells were transformed with the recovered DNA.1 μl of a 1/10 dilution of the isolated phage DNA was used in thetransformation. The transformation mixture was plated and plaques werescreened with oligonucleotides as previously described.

A large scale miniprep was performed on positive plaques which had beenincubated for approximately 7 hours in 2 mls of LB media. 25 mls of LBmedia was inoculated with 250 μl of freshly grown JM103 cells. Theculture was grown for 1 hour and inoculated with 100 μl of the 7 hourold plaque culture. The culture was then grown overnight, and thencentrifuged twice at 10,000 rpms for 10 minutes on a Sorvall RC-5B witha SS34 rotor to clear the supernatant. 3.5 ml of 20% PEG/2.5M NaCl wasadded to the culture and it was incubated for 5 hours at 4° C. Theculture was then centrifuged again as above for 10 minutes. Thesupernatant was discarded and the pellet was resuspended in 2 mls of TEbuffer. The pellet was then extracted with phenol, equilibrated with TE,extracted with phenol/chloroform once, extracted twice with CHCl₃ andonce with ether. 8M LiCl was added to attain a final concentration of0.8M. 3 volumes of ethanol were added and the solution left overnight toprecipitate the DNA present. The solution was centrifuged for 10 minutesat 10,000 rpm as previously described and rinsed with 70% ethanol. Theprecipitate was resuspended in 150 μl of 10 mM Tris (pH 7.4).

Positive colonies were identified by colony hybridization Grunstein andHogness, PNAS 72,3961 (1975)! and sequenced using dideoxy sequencing tofind the M13 constructs with the correct mutation. RF DNA was recoveredusing the alkaline lysis method of Maniatis et al. A 678 bp EcoRIfragment was isolated on a 0.8% preparative agarose gel and subclonedinto pA0804 and pA0811 (see Example V and VI, respectively). E. coliMC1061 cells were transformed with either of these two vectors asdescribed in Example I. Transformants containing the proper orientationwere identified by XbaI digestion of DNA minipreps (also as described inExample I). One transformant derived from pA0804 was called pTB06; apA0811 derived transformant was called pHB6.

Example V Construction of pA0803 and pA0804

pA0804 is a vector capable of site-specific disruption of the P.pastoris AOX1 locus. It contains the following elements: the AOX1promoter and transcription terminator separated by a unique EcoR1cloning site; the wild-type Pichia HIS4 gene; a genomic segment of DNAfrom the 3' end of the AOX1 locus downstream of the transcriptionterminator; sequences necessary for selection and replication in abacterial host. The components are arranged such that a restrictiondigest of the vector releases a DNA fragment containing the expressioncassette and selective marker whose ends are homologous to a continuousportion of the genome, the AOX1 locus, and can be stably inserted intothe chromosome during transformation.

pA0804 is a derivative of the hepatitis B surface antigen expressionplasmid pBSAGI5I (NRRL 18021). It was assembled in a pBR322-basedplasmid containing the following modifications. pBR322 was digested withEcoR1, followed by phenol extraction and ethanol precipitation. The endswere then filled in using Klenow polymerase. 2 μg of EcoR1 digestedpBR322 was incubated at room temperature for 15 minutes in a 25 μlreaction volume containing 50 mM Tris.Cl, pH 7.2, 10 mM MgSO₄, 100 μMdithiothrietol, 50 μg/ml bovine serum albumin, 100M dATP, 100 μM dTTP,and 1 unit of Klenow polymerase. Following phenol extraction and ethanolprecipitation, the DNA was ligated to reclose the plasmid and theligation reaction was used to transform E. coli MC1061. Transformantswere screened for the absence of the EcoRI site as well as the presenceof diagnostic sites such as PstI, PvuII and SalI. Such a plasmid wascalled pBR322-RI.

This plasmid was further modified to incorporate a BglII site at thePvuII site. The plasmid was digested with PvuII and phosphorylated BglIIlinkers (GAGATCTC) were added in a blunt end ligation. The excesslinkers were removed by overnight BglII digestion and the plasmid wasreclosed using T4 DNA ligase. The ligation reaction was used totransform E. coli MC1061 to ampicillin resistance. Transformants werecharacterized by restriction digestion with BglII to indicate thepresence of a BglII site and with a SalI/BglII digest indicating thatthe BglII site was at the former PvuII site. This plasmid was designatedpBR322 BglII-RI.

pA0804 and pA0811 were created by scavenging DNA fragments from pBSAGI5Iand assembling them in pBR322 BglII-RI. The 3' targeting segment of theAOX1 locus was removed from pBSAGI5I as a 700 bp BglII/XhoI fragment, ofwhich 50 ng were ligated to 5 ng of the parent plasmid which had beendigested with SalI and BglII. The ligation reaction was used totransform E. coli MC1061 to ampicillin resistance. Transformants werecharacterized by a BamHI/BglII digest of mini prep DNA as described inExample 1, such that an approximately 900 bp fragment was observed. Onesuch transformant was chosen and DNA was purified on large scale. Such aplasmid was named pA0801.

The plasmid pBSAGI5I was digested with ClaI and a 2.1 Kb fragmentcontaining the promoter-gene-terminator expression cassette wasisolated. The 2 μg of pA0801 was digested with ClaI and treated withalkaline phosphatase in a 30 μl reaction volume (1 U enzyme at 37° C.for 1 hour in 50 mM Tris HCl pH 9.0, 1 mM MgCl₂, 100 μM ZnCl₂, 1 mMspermidine). 50 ng of the ClaI fragment was ligated to 5 ng of thepA0801 vector and the ligation reaction was used to transform E. coliMC1061 to ampicillin resistance. These colonies were characterized byBglII digestion to ascertain that the fragment was inserted and was inthe correct orientation yielding a spectra of 2.3 and 2.7 Kb fragments.This single transformant was called pA0802.

The plasmid pA0802 was digested at the unique StuI site at the 3' end ofthe hepatitis B surface antigen gene. EcoRI linkers were phosphorylated,annealed, and ligated to the StuI digested plasmid. Excess linkers wereremoved by overnight EcoRI digestion. The EcoRI digestion also cuts atthe 5' end of the HBsAg structural gene, hence removing the gene. Uponreligation, the promoter and transcription terminator were joined by aunique EcoRI cloning site. Ampicillin resistant transformants werecharacterized again by BglII digestion and a transformant with thecorrect spectrum (2.3 & 2.1 Kb) was identified and called pA0803.

The plasmid pA0803 was digested with BamHI and the 2.7 Kb BglII fragmentfrom pYM10 (FIG. 8) was isolated by preparative agarose gelelectrophoresis and ligated to the BamHI-digested dephosphorylatedpA0803. pYM10 is a derivative of pYJ30 (NRRL B-15890) with the BamHIsite at 2959 destroyed!. Transformants were characterized by thepresence of an XbaI site yielding a fragment of 7.4 Kb and a BglIIspectrum of 2.3 and 5.1 Kb. This plasmid was called pA0804.

Example VI Construction of pA0811

A second related plasmid containing the Saccharomyces ARG4 gene insteadof the Pichia HIS4 gene was also constructed. One possible source of theARG4 gene is the 2.0 Kb HpaI fragment obtained from pYM25, a plasmid inan E. coli host, NRRL B-18015. This strain is available from theNorthern Regional Research Center of the United States Department ofAgriculture, Peoria, Ill.

The fragment was purified from a 0.8% preparative agarose gel. 500 ng ofthe fragment was ligated to 50 ng of BamHI digested, filled in pA0803(see Example V). The ligation reaction was used to transform E. coliMC1061 to ampicillin resistance. Transformants were characterized byBglII digestion, and the correct insert size was verified by agarose gelelectrophoresis. This plasmid was called pA0811.

Example VII Yeast DNA Miniprep

10⁴ cells/ml were grown in 5 ml YPD at 30° C. overnight and thenpelleted using a Damon IEC DPR600 clinical centrifuge at 3,000 rpm for 5minutes. The pellet was resuspended in 0.5 ml of 1M sorbitol, 0.1 ml0.5M EDTA, pH 7.5 and the sample transferred to a 1.5 ml microfuge tube.0.02 ml of 2.5 mg/ml Zymolyase 60,000 (Miles Laboratories) was added,and the sample was incubated at 37° C. for 60 minutes. The cells werepelleted using the microfuge for 1 minute at high speed, and resuspendedin 0.5 ml of 50 mM Tris.Cl, pH 7.4 and 20 mM EDTA. 0.05 ml of 10 % SDSwas added, the sample mixed, and incubated at 65° C. for 30 minutes. 0.2ml of 5M potassium acetate was added and the sample was incubated on icefor 60 minutes. The sample was again spun in a microfuge at high speedfor 5 minutes.

The supernatant was transferred to a fresh 1.5 ml microfuge tube and 1volume of isopropanol at room temperature was added. The sample wasmixed and allowed to sit at room temperature for 5 minutes, then spunvery briefly (10 seconds) in a microfuge at high speed. The supernatantwas poured off and the pellet air dried. After resuspending the pelletin 0.3 ml of 10 mM Tris.Cl, pH 7.4 and 1 mM EDTA, 15 μl of a 1 mg/mlsolution of pancreatic RNase was added, and the sample was incubated at37° C. for 30 minutes. 0.03 ml of 3M sodium acetate was added, thesample mixed, and 0.2 ml of isopropanol added. The sample was spun in amicrofuge at high speed to pellet the DNA. The supernatant was thenpoured off, the pellet dried and resuspended in 0.1-0.3 ml of 10 mMTris.Cl, pH 7.4 and 1 mM EDTA. (Note: Before using the DNA in arestriction digest, it may be necessary to spin the solution for 15minutes at high speed in the microfuge to remove any insoluble materialwhich may inhibit the digestion).

Example VIII Development of Mixed Particle Strains

Two mixed particle strains containing expression cassettes encoding theS (p24) and preS₂ (p31) forms of the Hepatitis B surface antigen wereconstructed as follows. Pichia pastoris PPF1 (arg4 his4) was transformedwith 1 μg of uncut pHB6 using the spheroplast transformation techniquedescribed by Cregg et al., Bio/Technology 5,479 (1987). (pHB6 is asubclone of pA0811 containing the S gene described in Example IV andVI). Transformants demonstrating arginine prototrophy were regeneratedon minimal media containing histidine and screened for the site ofintegration as follows.

DNA from these transformants and from wild type Pichia pastoris wasprepared as described in Example VII, digested with EcoR1 and subjectedto electrophoresis on 0.8% agarose. Southern blots of these DNAs wereperformed (Maniatis et al. 1983) and the filters hybridized with an AOX1specific probe (pPG4.0 NRRL#15868) or with a HIS4 specific probe (pYM4).pYM4 is described in Example I. The site of integration was determinedby comparing the spectrum of hybridization of a given transformant withthe wild type strain. Any alteration in the size of the wild type bandwas evidence of integration at that locus. The transformant containingan integration at the 5'-end of the AOX1 locus, and still containing awild type AOX1 gene as well as a HIS4 mutation, was called PPF1/pHB6.

This strain was then transformed with BglII-cut pTB05A (from Example II)as described above. Transformants demonstrating histidine prototrophywere regenerated on minimal media and screened for the "methanol slow"(Mut-) phenotype, which indicated integration of the AOX1 locus.Screening for the phenotype was performed in the following manner.

Transformants were pooled by scraping the surface of the plate in thepresence of sterile distilled water and sonicated at low output for 15seconds. They were subsequently diluted to an A₆₀₀ 0.1 and plated atdilutions of 10⁻³ and 10⁻⁴ in duplicate onto minimal plates containingglycerol as the carbon source, and incubated at 30° C. for 2-3 days.They were then replica-plated onto minimal plates to which 100 μl ofmethanol was added in the vapor phase. After a 24-hour incubation at 30°C., it was apparent that 10-20% of the transformants were growing moreslowly on methanol than the rest of the transformants. Ten of these slowgrowing colonies were then selected for further analysis. They werepicked from the minimal plate containing glycerol, grown in shake flasksas described in Example IX, and assayed for 22 nm--like particleactivity as described in Example XI. The transformants werecharacterized as described above for pHB6. One resulting strain wascalled PPF1/pTB012-1, and expressed one copy of both the p24 and p31proteins.

Another strain was identified which had integrated two copies of thepreS₂ expression cassette. It was called PPF1/pTB012-2 and it expressedtwo copies of the preS₂ gene and one copy of the S gene.

Particle expression levels are shown in Table 3.

                  TABLE 3                                                         ______________________________________                                                                    Particle Expression Levels                        Strain     Cassette Proteins                                                                              (AUSRIA ™)                                     ______________________________________                                        PPF1/pTB012-1                                                                            1 preS.sub.2                                                                           p31     ˜150-200                                               1 S      p24     μg particle/ml lysate                          PPF1/pTB012-2                                                                            2 preS.sub.2                                                                           p31     ˜150-200                                               1 S      p24     μg particle/ml lysate                          ______________________________________                                    

In addition, SDS/PAGE analysis and silver staining of a partiallypurified protein preparation indicated the presence of both p24 and p31.

Example IX Shake Flask Expression Studies

Prior to fermentation, all strains were grown in shake flasks as followsto ascertain expression levels. Routinely, a transformant was seededinto 0.67% yeast nitrogen base containing 2-5% glycerol and grownovernight at 30° C. into middle to late log phase. The cells were thencollected by centrifugation rising a Damon IEC DPR600 clinicalcentrifuge at 3,000 rpm for 5 minutes. The pellet was washed in sterilewater twice, then seeded at a density of 0.5 A₆₀₀ units/ml intophosphate buffered 0.67% YNB containing 1% methanol and grown for 4-6days at 30° C. with moderate shaking. At various times, aliquots of 50A₆₀₀ units were removed and stored at -20° C. Protein extracts wereprepared from these aliquots to be used for an AUSRIA™ (see Example XI)and Western blot analysis Towbin et al. PNAS 76, 4350 (1979)!. Antibodywas Lot #702106 from Calbiochem, used at a 1:1000 dilution. Aliquots ofcells (100 A600 units) were transferred to 13×100 mm borosilicateculture tubes and washed twice by centrifugation in a Sorvall ModelRC-5B at 12,000 rpm, 4° C. with 20 volumes of lysing buffer 0.5M NaCl₁,0.1 % Triton X-100 (^(w) /v), 1M phenylmethylsulfonyl fluoride and 10 mMsodium phosphate, pH 7.5)!. Cell samples were resuspended with 0.5 gramsof acid-washed glass beads (0.5 mm) plus 0.35 ml of lysing buffer, andagitated for eight, one-minute intervals at maximum speed using a vortexmixer. Between intervals, the mixture was cooled on ice for at least oneminute. After lysing was completed, the solution of broken cells wasremoved and the glass beads were washed with 0.35 ml of lysing buffer.The two solutions were then combined and centrifuged using the SorvallRC-5B at 13,000 rpm, 4° C., to remove cellular debris. Protein sampleswere then assayed by AUSRIA™ and by Western blot analysis. Proteinconcentration was determined by the Lowry method after TCAprecipitation.

Example X Fermentation Expression Studies

Fermentations were performed as follows. Five hundred ml of YeastNitrogen Base (YNB) +2% glycerol in a Fernbach Flask was inoculated froma seed culture or a minimal glucose plate of the culture. (Plates may bemaintained by monthly passage with no detectable strain deterioration).After one day of shaking at 200 rpm and 30° C., the inoculum was seededinto 7.5-liter minimal medium (Table 4) containing 480 g glycerol, 40 mgbiotin, and 40 ml trace salts solution (Table 5). The fermentor wasmaintained at 30° C. and pH 5.5 while the culture grew in batch modeuntil the glycerol was exhausted (about 24 hours). The pH was controlledby the addition of NH₃ gas. Glycerol exhaustion was noted by a sharpdecline in the CO₂ evolution and a sharp rise in the dissolved oxygen(or decrease in oxygen uptake rate). A methanol feed was initiated at 18ml/hr to bring the fermentor level up to ˜0.5% MeOH, and maintained atthis level. The flow rate was adjusted based on the actual methanolconsumption rate. Twenty ml aliquots of trace salts were added atapproximately two day intervals to maintain the methanol consumptionrate. The level of HBsAg increased for approximately 7-8 days on themethanol feed.

                  TABLE 4                                                         ______________________________________                                        Medium Composition (7.5-Liter)                                                ______________________________________                                        480        g            glycerol                                              40         mg           biotin                                                134        ml           H.sub.3 PO.sub.4 (85%)                                5.8        g            CaSO.sub.4.2H.sub.2 O                                 92         g            H.sub.2 SO.sub.4                                      75         g            MgSO.sub.4.7H.sub.2 O                                 21         g            KOH                                                   ______________________________________                                    

                  TABLE 5                                                         ______________________________________                                        IM.sub.1 Trace Salts Solution                                                 ______________________________________                                        Cupric Sulfate.5H.sub.2 O                                                                           0.06                                                    Potassium Iodide      0.08                                                    Manganese Sulfate.H.sub.2 O                                                                         0.30                                                    Sodium Molybdate      0.20                                                    Boric Acid            0.02                                                    Zinc Sulfate.H.sub.2 O                                                                              2.00                                                    Ferric Chloride.H.sub.2 O                                                                           4.8                                                     Sulfuric Acid         5.00 ml/liter                                           ______________________________________                                    

Example XI AUSTRIA™ RIA Protocol

The Abbott AUSRIA™ assay kit was used to measure the amount of HBsAgsynthesized by the Pichia production system. The antibody contained inthe kit binds to HBsAg particles, not HBsAg monomers. All dilutions weremade in 1.0% BSA, 0.02% Na Azide in phosphate buffered saline, pH 7.4.The procedure followed was essentially as outlined in the kitinstructions. The standard curve was prepared as follows.

    ______________________________________                                        Tube #   ng. in assay                                                                            Buffer (μl)                                                                           Positive Control (μl)                        ______________________________________                                        1-4      None NSB  none       200 buffer only                                 5-6      0.1       195         5                                              7-8      0.2       190         10                                              9-10    0.5       175         25                                             11-12    1.0       150         50                                             13-14    2.0       100        100                                             15-16    3.0        50        150                                             17-18    4.0       none       200                                             ______________________________________                                    

The wells of the microtiter dish were labeled as follows.

    ______________________________________                                               AA   BB        CC    DD                                                ______________________________________                                        1        1      2         3    4                                              2        5      6         7    8                                              3        9      10        11  12                                              4        13     14        15  16                                              5        17     18        19  20 and so on . . .                              ______________________________________                                    

The beads were first added to each well, followed by the buffer, andfinally the standard (positive control) or the diluted sample. Unknownswere diluted to obtain signals within the range of the standard curve.Estimates of sample concentrations in mg/ml were typically divided by0.02 to obtain the dilution to be used. Usually 100 μl of the sample wasadded to the well containing 100 μl of buffer. The wells were coveredand the tray gently tapped against the bench top. The samples were thenincubated overnight at room temperature to attain maximum bindingefficiency. The next morning each well was washed 4 times with deionizedwater using the Pentawash system provided by Abbott Labs. 200 μl of the¹²⁵ I anti-HBs were added to each well, the tray was gently tapped, andthen incubated in a 45° C. water bath for 1 hr. The beads were washed asbefore and counted. Concentrations of unknowns were determined from thestandard curve.

That which is claimed is:
 1. Antigenic Hepatitis B virus (HBV) particlescomprising HBV S and preS2 proteins made by a process whichcomprises:(a) transforming a Pichia pastoris strain with a firstexpression cassette comprising a structural gene for a hepatitis B virusS protein operably linked to a 5' regulatory region and a 3' terminationsequence obtainable from Pichia pastoris; and (b) transforming saidPichia pastoris strain with a second expression cassette comprising astructural gene for the hepatitis B virus preS₂ protein operably linkedto a 5' regulatory region and a 3' termination sequence obtainable fromPichia pastoris; and (c) culturing the resulting transformed Pichiapastoris strain under suitable conditions to obtain the production ofsaid HBV particles.
 2. Recombinantly produced HBV particles consistingof S and preS₂ proteins.
 3. Hepatitis B virus (HBV) particles comprisingS and preS₂ proteins produced in a methylotrophic yeast.